Small RNA Analysis in Sindbis Virus Infected Human HEK293 Cells

Andras Donaszi-Ivanov,Tamas Dalmay,Irina Mohorianu,Penny P Powell,Zach N Adelman

doi:10.1371/journal.pone.0084070

Andras Donaszi-Ivanov, Tamas Dalmay + Show 3 more

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https://doi.org/10.1371/journal.pone.0084070

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Journal: PloS one	Publication Date: Dec 31, 2013
Citations: 52	License type: CC BY 4.0

Affiliation: University of East Anglia

Abstract

IntroductionIn contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate. Since RNAi has a well-established role in controlling infection of the alphavirus Sindbis virus (SINV) in insects, we have used this virus to investigate the role of RNAi in SINV infection of human cells.ResultsSINV AR339 and TR339-GFP were adapted to grow in HEK293 cells. Deep sequencing of small RNAs (sRNAs) early in SINV infection (4 and 6 hpi) showed low abundance (0.8%) of viral sRNAs (vsRNAs), with no size, sequence or location specific patterns characteristic of Dicer products nor did they possess any discernible pattern to ascribe to a specific RNAi biogenesis pathway. This was supported by multiple variants for each sequence, and lack of hot spots along the viral genome sequence. The abundance of the best defined vsRNAs was below the limit of Northern blot detection. The adaptation of the virus to HEK293 cells showed little sequence changes compared to the reference; however, a SNP in E1 gene with a preference from G to C was found.Deep sequencing results showed little variation of expression of cellular microRNAs (miRNAs) at 4 and 6 hpi compared to uninfected cells. Twelve miRNAs exhibiting some minor differential expression by sequencing, showed no difference in expression by Northern blot analysis.ConclusionsWe show that, unlike SINV infection of invertebrates, generation of Dicer-dependent svRNAs and change in expression of cellular miRNAs were not detected as part of the Human response to SINV.

Highlights

In contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate
In plants, insects and nematodes, RNAi can serve as an innate immune response against viruses, as double stranded RNA (dsRNA) produced by viruses as intermediates of replication is processed by a RNA Induced Silencing Complex (RISC) into small interfering RNAs to target complementary viral mRNAs for destruction
In this study we looked for the presence of viral sRNAs (vsRNAs) during a time course of Sindbis virus (SINV) infection of human embryonic kidney 293 cells (HEK 293) and the changes in cellular miRNA profiles by high throughput sequencing

Summary

Introduction

In contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate. In plants, insects and nematodes, RNAi can serve as an innate immune response against viruses, as dsRNA produced by viruses as intermediates of replication is processed by a RNA Induced Silencing Complex (RISC) into small interfering RNAs (siRNAs) to target complementary viral mRNAs for destruction. It is still a matter of debate if RNAi plays a role in innate immunity in mammals [1,2] because the sRNAs were only isolated at low concentration [3] and it is unclear whether the observed fragments are DICER dependent or not [4]. An initial study using deep sequencing from infection of a broad range of animal cells with six different RNA and DNA viruses showed the existence of some vsRNAs and changes in host miRNA expression [3]

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